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Abstract：
Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) is a multifunctional scaffolding protein that interacts with receptors and ion transporters in its PDZ domains and with the ezrin-radixin-moesin (ERM) family of proteins in its COOH terminus. The role of NHERF1 in hepatocyte function remains largely unknown. We examine the distribution and physiological significance of NHERF1 and multidrug resistance-associated protein 2 (Mrp-2) in hepatocytes. A WT radixin binding site mutant (F355R) and NHERF1 PDZ1 and PDZ2 domain adenoviral mutant constructs were tagged with yellow fluorescent protein and expressed in polarized hepatocytes to study localization and function of NHERF1. Cellular distribution of NHERF1 and radixin was visualized by fluorescence microscopy. A 5-chloromethylfluorescein diacetate (CMFDA) assay was used to characterize Mrp-2 function. Similar to Mrp-2, WT NHERF1 and the NHERF1 PDZ2 deletion mutant were localized to the canalicular membrane. In contrast, the radixin binding site mutant (F355R) and the NHERF1 PDZ1 deletion mutant, which interacts poorly with Mrp-2, were rarely associated with the canalicular membrane. Knockdown of NHERF1 led to dramatically impaired CMFDA secretory response. Use of CMFDA showed that the NHERF1 PDZ1 and F355R mutants were devoid of a secretory response, while WT NHERF1-infected cells exhibited increased secretion of glutathione-methylfluorescein. The data indicate that NHERF1 interacts with Mrp-2 via the PDZ1 domain of NHERF1 and, furthermore, that NHERF1 is essential for maintaining the localization and function of Mrp-2.
摘要：
Na（）/H（）交换剂调节因子1（NHERF1）是一种多功能支架蛋白，可与PDZ结构域中的受体和离子转运蛋白相互作用，并与COOH末端的ezrin-radixin-moesin（ERM）蛋白家族相互作用。NHERF1在肝细胞功能中的作用仍然未知。我们研究了NHERF1和多药耐药相关蛋白2（Mrp-2）在肝细胞中的分布和生理意义。用黄色荧光蛋白标记WTradixin结合位点突变体（F355R）和NHERF1PDZ1和PDZ2结构域腺病毒突变体构建体，并在极化肝细胞中表达，以研究NHERF1的定位和功能。通过荧光显微镜观察NHERF1和radixin的细胞分布。使用5-氯甲基荧光素二乙酸酯(CMFDA)测定来表征Mrp-2功能。与Mrp-2相似，WTNHERF1和NHERF1PDZ2缺失突变体位于小管膜上。相比之下,radixin结合位点突变体（F355R）和NHERF1PDZ1缺失突变体，与Mrp-2相互作用较差，很少与小管膜相关。NHERF1的击倒导致CMFDA分泌反应严重受损。CMFDA的使用表明，NHERF1PDZ1和F355R突变体没有分泌反应，而WTNHERF1感染的细胞显示谷胱甘肽-甲基荧光素的分泌增加。数据表明NHERF1通过NHERF1的PDZ1结构域与Mrp-2相互作用，此外，NHERF1对于维持Mrp-2的定位和功能至关重要。
